CN115322163A - Di-methylamino-s-tetrazine compound as VEGFR-2 inhibitor and preparation and application thereof - Google Patents
Di-methylamino-s-tetrazine compound as VEGFR-2 inhibitor and preparation and application thereof Download PDFInfo
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- CN115322163A CN115322163A CN202210879091.5A CN202210879091A CN115322163A CN 115322163 A CN115322163 A CN 115322163A CN 202210879091 A CN202210879091 A CN 202210879091A CN 115322163 A CN115322163 A CN 115322163A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/08—Six-membered rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a diamyl amido symtetrazine compound and a preparation method and application thereof. The bis-methylamido-sym-tetrazine compound has the following structural formula (I), wherein R is methyl, ethyl, propyl, isobutyl, benzyl, 3-chlorobenzyl, 3,4-dimethoxybenzyl, 3-methylphenylaminomethyl, 3,4-dimethylphenylaminomethyl, 3-trifluoromethyl-4-chlorophenylaminomethyl, 3,4-dichlorophenylaminomethyl, 3-fluoro-4-chlorophenylaminomethyl, phenylaminomethyl, 2-methylphenylaminomethyl, 3,5-dimethylphenylaminomethyl, phenylamino, 3-methoxyphenylamino, 3,4-dimethoxyphenylamino, 2,5-difluorophenylamino, 2-methylphenylamino, 3-methylphenylamino, 4-methylphenylamino or 3,5-dimethylphenylamino. The invention provides said compounds orThe application of the pharmaceutically acceptable salt in the preparation of medicaments for treating or preventing VEGFR-2 mediated diseases or medicaments for inhibiting VEGFR-2 shows good inhibitory activity.
Description
Technical Field
The invention relates to a diformylamido-pyromellitic dianhydride compound, a preparation method thereof and application of the compound serving as a VEGFR-2 inhibitor in preparing medicaments for treating or preventing VEGFR-2 mediated diseases or VEGFR-2 inhibiting medicaments.
Background
The tetrazine compound has many better physical properties, spectral properties and higher reaction activities, and particularly some tetrazine derivatives with special structures have obvious antitumor activity and antiviral activity and can be used as pesticides and insecticides. For example, two varieties of pesticides (clofentezine and flutenzine) are on the market, and one variety of medicines (temozolomide, an anti-tumor medicine) is on the market.
In 1978, 3,6-dibenzynyl-hexahydro-1,2,4,5-tetrazine was reported to have antitumor activity (see eremev, a.v.; tikhomirova, d.a.; tyusheva, v.a.; liepens, f.khim. Geotsikl.soedin, 1978,753), which is the first reported potential antitumor activity of 1,2,4,5-tetrazine. Later, some 1,2,4,5-tetrazines were reported to have antitumor activity, such as 3,6-bis (2 '-hydroxy-5' -chlorophenyl) -1,2,4,5-tetrazine (see Rao, G. -W.; hu, W. -X.Bioorg.Med.chem.Lett.2006,16 (14), 3702), which has antitumor activity. Of course most 1,2,4,5-tetrazines do not have antitumor activity.
Disclosure of Invention
The first object of the present invention is to provide a novel class of bis-methylamidosym-tetrazines having inhibitory activity against VEGFR-2 mediated diseases and VEGFR-2.
The second purpose of the invention is to provide a preparation method of the diformylamido-tetrazine compound, which has the advantages of simple preparation method, easy operation, easily available raw materials, lower production cost and suitability for industrial application.
The third purpose of the invention is to provide the application of the diformylaminomethyltetrazine compound or the pharmaceutically acceptable salt thereof in preparing medicaments for treating or preventing VEGFR-2 mediated diseases or VEGFR-2 inhibiting medicaments, wherein the VEGFR-2 mediated diseases are cancers, the cancers are human lung cancers, human liver cancers or human breast cancers, and the cancer cells of the cancers are A549, huh7, MDA-MB-231 or HepG2 cells.
The technical solution adopted by the present invention is specifically explained below.
In a first aspect, the present invention provides a bis-methylamido-tetrazine compound having the following structural formula (i):
in the formula (I), R is methyl, ethyl, propyl, isobutyl, benzyl, 3-chlorobenzyl, 3,4-dimethoxybenzyl, 3-methylphenylaminomethyl, 3,4-dimethylphenylaminomethyl, 3-trifluoromethyl-4-chlorophenylaminomethyl, 3,4-dichlorophenylaminomethyl, 3-fluoro-4-chlorophenylaminomethyl, phenylaminomethyl, 2-methylphenylaminomethyl, 3,5-dimethylphenylaminomethyl, phenylamino, 3-methoxyphenylamino, 3,4-dimethoxyphenylamino, 2,5-difluorophenylamino, 2-methylphenylamino, 3-methylphenylamino, 4-methylphenylamino or 3,5-dimethylphenylamino.
Particularly preferred compounds of the present invention are those selected from the group consisting of:
in a second aspect, the invention provides a preparation method of a bis-methylamido-tetrazine compound shown as a formula (I), wherein the preparation method comprises the following steps:
(1) Adding triphosgene into an organic solvent A, dropwise adding an organic solvent A solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in a formula (II) and an alkaline catalyst a under the stirring condition at the temperature of-10-12 ℃, heating the reaction solution to room temperature after the dropwise adding, stirring and reacting for 0.5-50 hours at the room temperature, introducing nitrogen into the reaction system until tail gas absorption liquid (NaOH aqueous solution) does not generate white mist, and directly using the generated product for the next reaction;
(2) Adding a compound shown as a formula (III) and a basic catalyst B into an organic solvent B, stirring for dissolving, dropwise adding the reaction liquid obtained in the step (1) under the stirring condition of-10-12 ℃, heating the reaction liquid to room temperature after dropwise adding, stirring for reacting for 0.5-50 hours at room temperature, and separating and purifying the reaction liquid to obtain the bisamido homotetrazine compound shown as a formula (I);
the basic catalyst a and the basic catalyst b are one of the following: triethylamine, 4-Dimethylaminopyridine (DMAP), pyridine or sodium hydroxide;
in the formula (III), R is as defined in the formula (I).
The reaction for preparing the bis-methylamino-pyromellitic compound (I) is shown as the following reaction formula, and the following reaction formula is not reported in the literature:
further, the ratio of the amount of the compound (ii) to the amount of the substance charged with the basic catalyst a, the basic catalyst b, the triphosgene and the compound (iii) is 1: 0.1 to 3, and preferably the ratio of the amount of the compound (ii) to the amount of the substance charged with the basic catalyst a, the basic catalyst b, the triphosgene and the compound (iii) is 1: 0.1 to 2.
Further, the organic solvent A and the organic solvent B are independently selected from one of the following: dichloromethane, chloroform or toluene. The organic solvent a may be used in an amount capable of dissolving triphosgene, the compound (ii), and the basic catalyst a, and the organic solvent B may be used in an amount capable of dissolving the basic catalyst B and the compound (iii). It is preferable that the total volume of the organic solvent A is 3 to 18mL/mmol based on the amount of the compound represented by the formula (II), the volume of the organic solvent A for dissolving triphosgene is 1 to 10mL/mmol based on the amount of the compound represented by the formula (II), and the total volume of the organic solvent B is 2 to 20mL/mmol based on the amount of the compound represented by the formula (II).
Further, in the step (1), under the condition of stirring at-10 to 5 ℃, an organic solvent A solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in the formula (II) and a basic catalyst a is added dropwise.
Further, in the step (2), the reaction liquid after the reaction in the step (1) is dropwise added under the condition of stirring at-10 to 5 ℃.
Further, the reaction processes of the steps (1) and (2) are followed by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 0.5-20.
Further, the separation and purification in the step (2) adopts the following steps: and (3) after the reaction is finished, washing the reaction solution with water, separating out an organic phase, evaporating to remove the solvent, and carrying out column chromatography on residues to obtain the bis-methylamido-tetrazine compound shown in the formula (I).
Furthermore, the column chromatography is specifically performed by the following steps: taking the residue after the solvent is removed by evaporation, adding an organic solvent C into the residue in a single-mouth bottle, dissolving the residue to obtain a dissolved solution, adding column chromatography silica gel (preferably 300-400-mesh coarse pore (zcx.II) type column chromatography silica gel) into the dissolved solution in an amount which is 1-2 times the mass of the residue, uniformly mixing, removing the solvent by evaporation to obtain a mixture of the dried residue and the silica gel, loading the mixture into a column, and then loading the mixture into the column according to a volume ratio of 0.5-20: 1 as an eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 0.5-20; the organic solvent C is one of the following solvents: petroleum ether, dichloromethane, chloroform or ethyl acetate; the organic solvent C is used in an amount capable of dissolving the residue.
The organic solvent A, the organic solvent B and the organic solvent C in the invention are all indicated as organic solvents for reaction or column chromatography, letters in the invention do not refer to certain organic solvents in any specific sense, and the letters are used for convenience of expression and are used for distinguishing the organic solvents appearing in different steps. The organic solvent A is the same organic solvent, and the organic solvent A, B or C can be the same solvent or different solvents.
In a third aspect, the invention provides an application of the diformylaminomethyltetrazine compound shown in the formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing VEGFR-2 mediated diseases or a medicament for inhibiting VEGFR-2.
In a specific embodiment of the invention, the VEGFR-2 mediated disease is cancer. Such cancers include, but are not limited to: non-small cell lung cancer, lung adenocarcinoma, breast cancer, liver cancer. In a preferred embodiment, the cancer is VEGFR-2 mediated non-small cell lung cancer or breast cancer or liver cancer, and the cancer cells of the cancer are A549, huh7, MDA-MB-231 or HepG2 cells.
Preferably, the cancer cells are A549 cells, and the diamylamido-pyromellizine compound represented by the formula (I) is a compound (I2 a), (I2 b), (I2 c), (I2 d), (I2 e) or (I2 g).
Preferably, the cancer cell is Huh7 cell, and the dicarbamido-pyromellizine compound represented by formula (i) is compound (i 2 b), (i 2 c), (i 2 e), (i 3 d), or (i 3 i).
Preferably, the cancer cell is a HepG2 cell, and the bisamido-homotetrazine compound represented by the formula (I) is a compound (I2 b), (I2 c) or (I3 d).
The term "pharmaceutically acceptable" refers herein to: the compounds are compatible chemically and/or toxicologically with the other ingredients comprising the formulation and/or with the human or mammal with which the disease or condition is being prevented or treated.
The term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic or organic acid addition salts of the compounds of the present invention. See, for example, S.M.Berge et al, "Pharmaceutical Salts", J.pharm.Sci.1977, 66,1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, or the like; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) -benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, diglucosic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, and the like.
Compared with the prior art, the invention has the following beneficial effects: (1) Provides a novel 1,2,4,5-tetrazine compound with good anti-cancer (especially human lung cancer, human liver cancer or human breast cancer) activity; (2) The preparation method of the 1,2,4,5-tetrazine compound is provided, the preparation method is simple, easy to operate, easy to obtain raw materials, low in production cost, suitable for practicality and expected to be applied to preparation of medicines for preventing or treating tumor diseases; (3) Provides the application of a novel 1,2,4,5-tetrazine compound or a pharmaceutically acceptable salt thereof in preparing medicaments for treating or preventing VEGFR-2 mediated diseases or VEGFR-2 inhibiting medicaments.
Detailed Description
The invention is further illustrated by reference to specific examples, which are intended to illustrate the invention, but not to limit it in any way.
3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) by Synthetic Communications,2003,33 (16), 2769-2775.
The following compounds (III 1) were prepared by the methods described in ChemMedChem,2019,14 (16), 1514-1527, european Journal of Medicinal Chemistry,2020,208, 112ACS Medicinal Chemistry letters,2012,3 (3), 232-237 ACS catalysis,2016,6 (7), 4189-4194, journal of Medicinal Chemistry,49 (17), 2006, 5080-5092.
Reference is made to the preparation of the following compounds (III 3) (Bioorganic & Medicinal Chemistry,2016,24 (18), 4241-4245, journal of Enzyme Inhibition and Medicinal Chemistry,2019,34 (1), 1573-1589.
The compound (III 2) is prepared by the following reaction formula, namely aniline compound and 2-chloro-N- (3-nitrophenyl) acetamide react under the action of triethylamine to generate intermediate product, the intermediate product reacts under the action of active iron powder to generate the compound (III 2), and R in the reaction formula 1 The structure is shown in detail in the structure of a compound (III 2), and the raw materials of 2-chloro-N- (3-nitrophenyl) acetamide and aniline compounds in the reaction formula are obtained by market:
the specific structure of the compound (III 2) is as follows:
example 1: preparation of Compound (III 2 f)
10.8g (50 mmol) of 2-chloro-N- (3-nitrophenyl) acetamide, 4.7g (50 mmol) of aniline, 5.1g (50 mmol) of triethylamine and 300.00mL of isopropanol are sequentially added into a three-neck flask, magnetic stirring is carried out, reflux reaction is carried out for 6 hours, organic solvent is evaporated, 150mL of dilute hydrochloric acid with pH of 1 is added, ethyl acetate is extracted (100 mL multiplied by 3), organic phases are combined, anhydrous magnesium sulfate is dried, filtration is carried out, organic solvent is evaporated, and 9.4g of N- (3-nitrophenyl) -2- (phenylamino) acetamide is obtained.
8.2g (30 mmol) of N- (3-nitrophenyl) -2- (phenylamino) acetamide, 200mL of ethanol, 75mL of distilled water and 10mL of glacial acetic acid were added in this order to a three-necked flask, and stirred magnetically, and then 1.0g (18 mmol) of active iron powder was added thereto, followed by reflux reaction for 5 hours. The mixture was filtered while hot, the filter cake was washed with ethyl acetate (80 mL. Times.3), the organic phases were combined, the solvent was evaporated off and the residue was taken up in saturated NaHCO 3 The aqueous solution was adjusted to pH 10, extracted with ethyl acetate (120 mL. Times.3), the organic phases were combined, dried over anhydrous magnesium sulfate, filtered and the solvent was distilled off to give 4.5g of the product (III 2 f).
Preparation of Compounds (III 2 a) to (III 2 e), (III 2 g) to (III 2 h): the aniline compounds were changed to prepare compounds (III 2 a) to (III 2 e), (III 2 g) to (III 2 h) by the method of example 1, and the details thereof are omitted.
Example 2: preparation of Compound (I1 a)
Dissolving 0.297g (1.0 mmol) of triphosgene in 10mL of chloroform, dropwise adding 20mL of chloroform solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.122g (1.0 mmol) of DMAP under magnetic stirring at the temperature of 10 ℃, heating the reaction solution to room temperature after completing the dropwise adding, reacting for 50 hours at the room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:2 is used as a developing agent), introducing nitrogen into the reaction system, and absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution) until no white mist is generated in the tail gas absorbing solution, and the generated product is directly used for the next reaction.
Dissolving 1.502g (10.0 mmol) of compound (III 1 a) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution after the previous step under the condition of magnetic stirring at-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 10 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:2), washing the reaction solution with (50 mL multiplied by 3) water, separating an organic phase, distilling off the solvent, and carrying out column chromatography on the residue, namely adding 10mL of petroleum ether solution into the residue after distilling off the solventDissolving the solvent with the solvent to obtain a solution, adding 1.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel) into the solution, uniformly mixing, evaporating the solvent to obtain a mixture of a dried residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 1:2 (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:2), collecting eluent containing the compound shown in the formula (I1 a) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (I1 a), with the yield of 33% (calculated on the basis of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine substance, except example 5, example 16 and example 28, the melting point is 198-200 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.96(s,1H),9.29(s,1H),8.82(s,1H),7.86(s,1H),7.33-7.31(m,1H),7.22-7.18(m,2H),2.19(s,3H),2.03(s,3H),1.94(s,3H).
Example 3: preparation of Compound (I1 a)
Dissolving 8.903g (30.0 mmol) of triphosgene in 100mL of dichloromethane, dropwise adding 80mL of dichloromethane solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.373g (30.0 mmol) of pyridine under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 0.5 hour at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20).
Dissolving 4.505g (30.0 mmol) of compound (III 1 a) and 2.373g (30.0 mmol) of pyridine in 200mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 0.5 hour, washing the reaction solution with (50 mL multiplied by 3) water, separating an organic phase, evaporating the solvent, and carrying out column chromatography, namely adding 10mL of dichloromethane solvent into the residue obtained after the solvent is evaporated to dissolve the residue, thus obtaining the productObtaining a dissolved solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel) into the dissolved solution, mixing uniformly, evaporating the solvent to obtain a mixture of a dried residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 20:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20. 1 H NMR as in example 2.
Example 4: preparation of Compound (I1 a)
5.935g (20.0 mmol) triphosgene is dissolved in 40mL of toluene, 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.024g (20.0 mmol) of triethylamine is added dropwise under magnetic stirring at 0 ℃, the reaction solution is warmed to room temperature after completion of the addition, the reaction is carried out for 3 hours at room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10 is used as a developing agent), nitrogen is introduced into the reaction system, and gas is absorbed by a tail gas absorption device (the tail gas absorption solution is a 10 NaOH aqueous solution), until no white mist is generated in the tail gas absorption solution, and the generated product is directly used for the next step reaction.
Dissolving 3.004g (20.0 mmol) of the compound (III 1 a) and 2.024g (20.0 mmol) of triethylamine in 50mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 8 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10:1, taking a mixed solution of petroleum ether and ethyl acetate as an eluent, and washingAnd (3) performing TLC (a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10 as a developing solvent) tracing detection, collecting eluent containing the compound shown in the formula (I1 a) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (I1 a), wherein the yield is 58%, and the melting point is 198-200 ℃. 1 H NMR as in example 2.
Example 5: preparation of Compound (I1 a)
Dissolving 1.484g (5.0 mmol) of triphosgene in 20mL of toluene, dropwise adding 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 12 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next step of reaction.
Dissolving 0.150g (1.0 mmol) of compound (III 1 a) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 50 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of chloroform solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a dissolved solution, then adding 0.25 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 5: eluting with mixed solution of petroleum ether and ethyl acetate as eluent, detecting by TLC (mixed solution of petroleum ether and ethyl acetate at 5:1 as developing solvent), collecting eluate containing compound shown in formula (I1 a) according to TLC detection, evaporating solvent from the collected eluate, and drying to obtain white solid product, i.e. compound (I1 a), with yield of 50% (based on compound III 1 a)Mass basis), melting point 198-200 deg.C. 1 H NMR as in example 2.
Example 6: preparation of Compound (I1 a)
Dissolving 1.484g (5.0 mmol) of triphosgene in 20mL of toluene, dropwise adding 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 12 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next step of reaction.
Dissolving 1.502g (10.0 mmol) of the compound (III 1 a) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane under the condition of magnetic stirring at-5 ℃, dropwise adding the reaction solution obtained in the previous step, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 9 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of chloroform solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 2.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), eluent containing the compound shown as the formula (I1 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 a), is obtained after drying, the yield is 46 percent, and the melting point is 198-200 ℃. 1 H NMR as in example 2.
Example 7: preparation of Compound (I1 b)
2.968g (10.0 mmol) triphosgene is dissolved in 40mL of toluene, 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine is added dropwise under the condition of magnetic stirring at 0 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 8 hours at the room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is 10 NaOH aqueous solution), until no white fog is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 1.642g (10.0 mmol) of compound (III 1 b) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 9 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL multiplied by 3) water, separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the mixture to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture to 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown as the formula (I1 b) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 b), is obtained after drying, the yield is 66 percent, and the melting point is 212-214 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.86(s,1H),9.28(s,1H),8.80(s,1H),7.89(s,1H),7.35-7.32(m,1H),2.30(q,J＝7.5Hz,2H),2.18(s,3H),1.93(s,3H),1.07(t,J＝7.6Hz,3H).
Example 8: preparation of Compound (I1 c)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 1.782g (10.0 mmol) of compound (III 1 c) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 9 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL × 3) water, separating out an organic phase, evaporating the solvent, performing residue column chromatography, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture is 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown in the formula (I1 c) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 c), is obtained after drying, the yield is 60 percent, and the melting point is 237-239 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.86(s,1H),9.28(s,1H),8.81(s,1H),7.90(s,1H),7.34-7.32(m,1H),7.22-7.13(m,2H),2.27(t,J＝7.3Hz,2H),2.19(s,3H),1.94(s,3H),1.64-1.57(m,2H),0.91(t,J＝7.4Hz,3H).
Example 9: preparation of Compound (I1 d)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 1.923g (10.0 mmol) of compound (III 1 d) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 10 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, performing residue column chromatography, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the residue to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown as the formula (I1 d) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 d) is obtained after drying, the yield is 65 percent, and the melting point is 224-226 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.27(s,1H),9.20(s,1H),8.79(s,1H),7.91(s,1H),7.34-7.32(m,1H),7.22-7.15(m,2H),2.19(s,3H),1.93(s,3H),1.21(s,9H)； 13 C NMR(125MHz,DMSO-d 6 )δ176.5,154.9,149.2,141.5,139.8,138.6,128.5,115.4,114.9,112.1,55.0,27.3,17.9,15.7.
Example 10: preparation of Compound (I1 e)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.263g (10.0 mmol) of compound (III 1 e) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column in a volume ratio of 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown as the formula (I1 e) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 e) is obtained after drying, the yield is 66 percent, and the melting point is 202-204 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ10.18(s,1H),9.28(s,1H),8.84(s,1H),7.94(s,1H),7.38-7.30(m,5H),7.26-7.23(m,1H),7.17-7.21(m,2H),3.63(s,2H),2.20(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ170.3,169.1,154.8,149.1,141.3,139.4,138.7,136.0,129.1,128.76,128.3,126.5,114.8,110.6,43.3,17.7,15.5.
Example 11: preparation of Compound (I1 f)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.607g (10.0 mmol) of compound (III 1 f) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after completing dropwise addition, reacting at room temperature for 13 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL multiplied by 3) water, separating out an organic phase, distilling off the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after distilling off the solvent to dissolve the residue to obtain a dissolved solution, then adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, distilling off the solvent to obtain a mixture of dried residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown in the formula (I1 f) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 f), is obtained after drying, the yield is 65 percent, and the melting point is 240-242 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ10.21(s,1H),9.29(s,1H),8.85(s,1H),7.93(s,1H),7.40(s,1H),7.43-7.27(m,4H),7.19(d,J＝6.7Hz,2H),3.66(s,2H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ169.5,154.9,149.3,148.7,147.8,141.5,139.7,138.9,128.9,128.6,121.3,115.0,114.1,113.3,112.1,110.9,43.1,17.9,15.7.
Example 12: preparation of Compound (I1 g)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.863g (10.0 mmol) of compound (III 1 g) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 14 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 4.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown as the formula (I1 g) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 g) is obtained after drying, the yield is 58 percent, and the melting point is 218-219 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ10.09(s,1H),9.28(s,1H),8.83(s,1H),7.91(s,1H),7.35-7.32(m,1H),7.2(dd,J＝8.5,3.5Hz,2H),6.95(s,1H),6.90(s,1H),6.85-6..83(m,1H),3.53(s,2H),2.19(s,3H),1.94(s,3H).
Example 13: preparation of Compound (I2 a)
Dissolving 0.297g (1.0 mmol) of triphosgene in 10mL of chloroform, dropwise adding 20mL of chloroform solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.122g (1.0 mmol) of DMAP under the condition of magnetic stirring at the temperature of-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 50 hours at the room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:2), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 2 a) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution after the previous step of reaction under the condition of magnetic stirring at-10 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 10 hours (the reaction process is detected by tracking TLC, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:2), washing the reaction solution with (50 mL x 3) water, separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of petroleum ether solvent into the residue after the solvent is evaporated, dissolving the residue to obtain a dissolved solution, adding 2.0 g of silica gel (silica gel for column chromatography of 300-400 mesh crude pore (zcx.II) type to the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, loading the mixture into a column, and then loading the mixture into a column in a volume ratio of 1:2 as eluent, eluting, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:2), collecting eluent containing the compound shown in the formula (I2 a) according to TLC detection, evaporating the collected eluent to remove the solvent, and drying to obtain a white solid product, namely the compound (I2 a), wherein the yield is 31%, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.92(s,1H),9.28(s,1H),8.84(s,1H),7.90(s,1H),7.39-7.32(m,1H),7.19(d,J＝5.2Hz,2H),6.97(t,J＝7.7Hz,1H),6.43-6.36(m,2H),5.88(t,J＝6.2Hz,1H),3.82(d,J＝6.2Hz,2H),2.18(s,3H),1.93(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ169.8,155.2,149.4,148.5,141.7,139.2,139.0,138.2,129.1,117.8,115.4,114.4,113.4,111.2,109.9,47.6,21.6,18.0,15.7.
Example 14: preparation of Compound (I2 a)
Dissolving 8.903g (30.0 mmol) of triphosgene in 100mL of dichloromethane, dropwise adding 80mL of dichloromethane solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.373g (30.0 mmol) of pyridine under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 0.5 hour at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20).
Dissolving 7.659g (30.0 mmol) of compound (III 2 a) and 2.373g (30.0 mmol) of pyridine in 200mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 0.5 hour, wherein the reaction process is detected by TLC (TLC tracing, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20:1 as eluent, eluting, tracking and detecting by TLC (a developing solvent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20. 1 H NMR and 13 c NMR was the same as in example 13.
Example 15: preparation of Compound (I2 a)
Dissolving 5.935g (20.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.024g (20.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 3 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next step reaction.
5.106g (20.0 mmol) of a compound were dissolved in 50mL of tolueneAdding the product (III 2 a) and 2.024g (20.0 mmol) of triethylamine into the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the completion of the addition, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, and the developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracing detection is carried out (the developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 10: 1), eluent containing the compound shown in the formula (I2 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 a) is obtained after drying, the yield is 47%, and the melting point is more than 300 ℃. 1 H NMR and 13 c NMR was the same as in example 13.
Example 16: preparation of Compound (I2 a)
Dissolving 1.484g (5.0 mmol) of triphosgene in 20mL of toluene, dropwise adding 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 12 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next step of reaction.
Dissolving 0.255g (1.0 mmol) of compound (III 2 a) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, and reacting at room temperature for 50 hours (the reaction process adopts TLC tracking detection, and the developing agents are petroleum ether and ethyl acetate with the volume ratio of 5:1Ester mixed solution), washing the reaction solution with (50 mL × 3) water, separating out an organic phase, evaporating to remove the solvent, performing column chromatography on the residue, namely adding 10mL of chloroform solvent into the residue after the solvent is evaporated to dissolve the residue to obtain a dissolved solution, adding 0.4 g of silica gel (300-400 meshes of coarse pore (zcx.ii) type column chromatography silica gel) into the dissolved solution, uniformly mixing, evaporating to remove the solvent to obtain a mixture of the dried residue and the silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), eluent containing the compound shown in the formula (I2 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 a) is obtained after drying, the yield is 52 percent (calculated by the substance of the compound III 2 a), and the melting point is more than 300 ℃. 1 H NMR and 13 c NMR was the same as in example 13.
Example 17: preparation of Compound (I2 a)
1.484g (5.0 mmol) of triphosgene is dissolved in 20mL of toluene, 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide is dropwise added under the condition of magnetic stirring at 5 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 12 hours at room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is a 10 NaOH aqueous solution), until no white mist is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 2 a) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at-5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 9 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1), washing the reaction solution with (50 mL multiplied by 3) water, separating an organic phase, removing the solvent by evaporation, carrying out column chromatography on residues, namely adding 10mL of chloroform solvent into the residues after removing the solvent by evaporation to dissolve the residues to obtain a dissolved solution, and then adding 10mL of chloroform solvent into the dissolved solution to obtain a dissolved solutionAdding 2.0 g of silica gel (300-400 meshes of coarse (zcx.II) type column chromatography silica gel), uniformly mixing, evaporating to remove the solvent to obtain a mixture of a dry residue and the silica gel, filling the mixture into a column, and then mixing the mixture in a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), eluent containing the compound shown in the formula (I2 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 a), is obtained after drying, the yield is 43 percent, and the melting point is more than 300 ℃. 1 H NMR and 13 c NMR was the same as in example 13.
Example 18: preparation of Compound (I2 b)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.693g (10.0 mmol) of compound (III 2 b) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 10 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column with a volume ratio of 6:1, eluting with mixed solution of petroleum ether and ethyl acetate, and detecting by TLC (The volume ratio of the developing solvent is 6:1, collecting the eluate containing the compound represented by the formula (i 2 b) according to TLC detection, evaporating the solvent from the collected eluate, and drying to obtain a white solid product, i.e., the compound (i 2 b), with a yield of 62% and a melting point of > 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.87(s,1H),9.27(s,1H),8.83(s,1H),7.89(s,1H),7.38-7.13(m,3H),6.84(d,J＝8.1Hz,1H),6.46-6.26(m,2H),5.71(d,J＝8.4Hz,1H),3.79(d,J＝6.5Hz,2H),2.18(s,3H),2.10(s,3H),2.06(s,3H),1.93(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ169.5,154.8,149.1,146.3,141.3,139.0,138.8,136.2,129.8,128.7,123.8,114.9,114.2,113.9,110.6,109.7,47.6,19.8,18.3,17.7,15.5.
Example 19: preparation of Compound (I2 c)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 3.437g (10.0 mmol) of compound (III 2 c) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 15 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column according to a volume ratio6:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 c) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 c), is obtained after drying, the yield is 51 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.93(s,1H),9.24(s,1H),8.83(s,1H),7.50(s,4H),7.14(t,J＝6.7Hz,1H),7.09(dd,J＝6.4,2.5Hz,1H),6.58-6.55(m,1H),6.23(d,J＝6.5Hz,1H),3.85(d,J＝6.4Hz,2H),2.17(s,3H),1.93(s,3H).； 13 C NMR(125MHz,DMSO-d 6 )δ168.8,155.2,149.6,148.2,141.8,139.5,139.3,132.4,129.2,127.1,124.7,122.5,116.8,116.3,115.5,114.5,111.2,47.0,18.2,16.0.
Example 20: preparation of Compound (I2 d)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 3.102g (10.0 mmol) of compound (III 2 d) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 14 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL multiplied by 3) water, separating out an organic phase, evaporating the solvent, carrying out residue column chromatography, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the residue to obtain a dissolved solution, then adding 4.0 g of silica gel (300-400 meshes of coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, and evaporating the solvent to obtain dry residueMixture of the retentate and silica gel, packing the mixture on a column and then mixing the mixture in a volume ratio of 6:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 d) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 d) is obtained after drying, the yield is 65 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.96(s,1H),9.27(s,1H),8.84(s,1H),7.91(s,1H),7.36(s,1H),7.20(d,J＝5.0Hz,2H),6.99(t,J＝8.2Hz,1H),6.20-6.15(m,2H),5.99(t,J＝6.3Hz,1H),3.67(d,J＝6.4Hz,2H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ168.9,155.2,149.6,149.1,141.8,139.5,139.3,131.7,130.9,129.3,117.4,115.5,114.5,113.6,113.2,111.2,47.1,18.2,16.0.
Example 21: preparation of Compound (I2 e)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.937g (10.0 mmol) of compound (III 2 e) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 14 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after evaporating the solvent to dissolve the residue to obtain a dissolved solution, and then adding 5.0 g of silica gel (300-400 meshes of coarse pores (zc) into the dissolved solutionx.II) type column chromatography silica gel), mixing, evaporating the solvent to obtain a mixture of dry residue and silica gel, loading the mixture into a column, and then mixing the dried residue and the silica gel in a volume ratio of 6:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 e) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 e) is obtained after drying, the yield is 77 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.99(s,1H),9.28(s,1H),8.84(s,1H),7.93(s,1H),7.28-7.26(m,1H),7.23(s,1H),7.21-7.18(m,3H),7.16(s,1H),7.15(s,1H),4.32(s,2H),2.18(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ168.9,159.3,157.4,155.3,149.6,141.8,139.5,139.3,130.8,129.2,115.5,114.5,111.2,110.0,107.6,100.3,47.2,18.2,16.0.
Example 22: preparation of Compound (I2 f)
2.968g (10.0 mmol) triphosgene is dissolved in 40mL of toluene, 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine is added dropwise under the condition of magnetic stirring at 0 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 8 hours at the room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is 10 NaOH aqueous solution), until no white fog is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 2.413g (10.0 mmol) of compound (III 2 f) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after the dropwise adding, reacting for 10 hours at room temperature (the reaction process is tracked and detected by TLC, a developer is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution by (50 mL multiplied by 3) water, separating an organic phase, distilling off the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after the solvent is distilled off to dissolve the residue to obtain a dissolved solution, and then adding 10mL of ethyl acetate solvent into the dissolved solution to obtain a solution3.0 g of silica gel (300-400 mesh coarse pore (zcx.ii) type column chromatography silica gel), mixing, evaporating the solvent to obtain a mixture of a dried residue and silica gel, loading the mixture into a column, and then mixing the dried residue and silica gel in a volume ratio of 6:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 f) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 f) is obtained after drying, the yield is 57 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.96(s,1H),9.27(s,1H),8.84(s,1H),7.91(s,1H),7.38-7.27(m,1H),7.19(d,J＝5.1Hz,2H),7.09(t,J＝7.9Hz,2H),6.58-6.53(m,3H),5.97(s,1H),3.84(d,J＝6.0Hz,2H),2.18(s,3H),1.93(s,3H).
Example 23: preparation of Compound (I2 g)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 2 g) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 10 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL multiplied by 3) water, separating an organic phase, evaporating the solvent, carrying out column chromatography on residues, namely adding 10mL of ethyl acetate solvent into the residues after solvent evaporation to dissolve the residues to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, and evaporating the solvent to obtain the dry productIs loaded onto a column and then the mixture is mixed with silica gel in a volume ratio of 6:1 is used as eluent to elute, TLC tracking detection (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 g) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and white solid product, namely the compound (I2 g) is obtained after drying, the yield is 59 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ10.17(s,1H),9.92(s,1H),7.91(s,1H),7.38(s,1H),7.21-7.17(m,2H),7.09(t,J＝7.9Hz,2H),6.58-6.54(m,3H),5.99-5.95(m,1H),3.84(d,J＝6.2Hz,2H),2.18(s,3H),2.03(s,3H),1.93(s,3H).
Example 24: preparation of Compound (I2 h)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.693g (10.0 mmol) of compound (III 2 h) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column with a volume ratio of 6:1 Petroleum ether and acetic acidEluting with ethyl ester mixed solution as eluent, detecting by TLC (mixed solution of petroleum ether and ethyl acetate at 6:1 as developing solvent), collecting eluate containing compound shown in formula (I2 h) according to TLC detection, evaporating solvent from the collected eluate, and drying to obtain white solid product, i.e. compound (I2 h), with yield of 57% and melting point of more than 300 deg.C. 1 H NMR(500MHz,DMSO-d 6 )δ9.90(s,1H),9.29(s,1H),8.85(s,1H),7.90(s,1H),7.35(t,J＝2.5Hz,1H),7.20(s,1H),7.19(s,1H),6.24-6.21(m,3H),5.78(dd,J＝7.2,2.4Hz,1H),3.80(d,J＝6.2Hz,2H),2.19(s,3H),2.14(s,6H),1.94(s,3H).
Example 25: preparation of Compound (I3 a)
Dissolving 0.297g (1.0 mmol) of triphosgene in 10mL of chloroform, dropwise adding 20mL of chloroform solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.122g (1.0 mmol) of DMAP under the condition of magnetic stirring at the temperature of-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 50 hours at the room temperature (the reaction process adopts TLC tracking detection, and a developing agent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:2), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next reaction.
Dissolving 2.273g (10.0 mmol) of compound (III 3 a) and 0.122g (1.0 mmol) of DMAP in 60mL of chloroform, dropwise adding the reaction solution after the previous step reaction under the condition of magnetic stirring at-10 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 10 hours at room temperature (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:2), washing the reaction solution with (50 mL × 3), separating an organic phase, removing the solvent by evaporation, carrying out column chromatography on the residue, namely adding 10mL of petroleum ether solvent into the residue after removing the solvent by evaporation to dissolve the residue to obtain a dissolved solution, then adding 2.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column silica gel chromatography into the dissolved solution by evaporation, uniformly mixing, removing the solvent by evaporation to obtain a mixture of dried residue and the silica gel, loading the mixture into a column, and then loading the mixture in a volume ratio of 1:2, eluting with mixed solution of petroleum ether and ethyl acetate as eluent, and detecting by TLC (the developing solvent is petroleum 1:2 by volume ratio)Ether and ethyl acetate mixed solution), collecting eluent containing the compound shown in the formula (I3 a) according to TLC detection, evaporating the solvent from the collected eluent, and drying to obtain a white solid product, namely the compound (I3 a), wherein the yield is 52 percent, and the melting point is 221-223 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.27(s,1H),8.82(s,1H),8.69(s,1H),8.58(s,1H),7.76(s,1H),7.47-7.41(m,2H),7.31-7.23(m,2H),7.21-7.10(m,3H),6.99-6.92(m,1H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ155.0,152.7,149.4,141.6,140.2,139.9,139.1,129.1,129.0,122.0,118.4,113.7,113.1,109.8,17.9,15.7.HRMS(ESI)m/z[M+Na] + calcd for C 18 H 19 N 7 NaO 2 :388.1498,found:388.1490.
Example 26: preparation of Compound (I3 a)
Dissolving 8.903g (30.0 mmol) of triphosgene in 100mL of dichloromethane, dropwise adding 80mL of dichloromethane solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.373g (30.0 mmol) of pyridine under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after the dropwise addition, reacting at room temperature for 0.5 hour (the reaction process adopts TLC tracking detection, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20 as a developing agent), introducing nitrogen into the reaction system, and absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution) until no white mist is generated in the tail gas absorbing solution, and directly using the generated product in the next step reaction.
Dissolving 6.818g (30.0 mmol) of compound (III 3 a) and 2.373g (30.0 mmol) of pyridine in 200mL of dichloromethane, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 12 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 0.5 hour, wherein the reaction process is detected by TLC (TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 20Packing the mixture into a column, and then mixing the mixture in a volume ratio of 20: the mixed solution of petroleum ether and ethyl acetate of 1 is used as eluent to elute, TLC tracking detection is carried out (a developing solvent is the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 20. 1 H NMR、 13 C NMR and HRMS were the same as in example 25.
Example 27: preparation of Compound (I3 a)
Dissolving 5.935g (20.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 2.024g (20.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 3 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is a 10 NaOH aqueous solution), and generating no white mist in the tail gas absorbing solution and directly using the generated product for the next step reaction.
Dissolving 4.545g (20.0 mmol) of compound (iii 3 a) and 2.024g (20.0 mmol) of triethylamine in 50mL of toluene, dropwise adding the reaction solution obtained in the previous step under magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 8 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 10): 1 as eluent, eluting, tracking and detecting by TLC (the developing solvent is a mixed solution of petroleum ether and ethyl acetate with the volume ratio of 10The eluent is collected, the solvent is evaporated and dried to obtain a white solid product, namely the compound (I3 a), the yield is 67 percent, and the melting point is 221-223 ℃. 1 H NMR、 13 C NMR and HRMS were the same as in example 25.
Example 28: preparation of Compound (I3 a)
1.484g (5.0 mmol) of triphosgene is dissolved in 20mL of toluene, 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide is dropwise added under the condition of magnetic stirring at 5 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 12 hours at room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is a 10 NaOH aqueous solution), until no white mist is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 0.227g (1.0 mmol) of compound (III 3 a) and 0.200g (5.0 mmol) of sodium hydroxide in 20mL of chloroform, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 5 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 50 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of chloroform solvent into the residue after solvent evaporation, dissolving the chloroform solvent to obtain a dissolved solution, adding 0.4 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture to 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), eluent containing the compound shown in the formula (I3 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 a), is obtained after drying, the yield is 58 percent (based on the substance of the compound III 3 a), and the melting point is 221-223 ℃. 1 H NMR、 13 C NMR and HRMS were the same as in example 25.
Example 29: preparation of Compound (I3 a)
1.484g (5.0 mmol) of triphosgene is dissolved in 20mL of toluene, 50mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 0.200g (5.0 mmol) of sodium hydroxide is dropwise added under the condition of magnetic stirring at 5 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 12 hours at room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is a 10 NaOH aqueous solution), until no white mist is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 2.273g (10.0 mmol) of compound (III 3 a) and 1.012g (10.0 mmol) of triethylamine in 60mL of dichloromethane, dropwise adding the reaction solution after the previous step reaction under the condition of magnetic stirring at-5 ℃, heating the reaction solution to room temperature after dropwise addition, reacting for 9 hours at room temperature (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 5:1), washing the reaction solution with (50 mL × 3), separating an organic phase, performing column chromatography on the residue after solvent evaporation, namely adding 10mL of chloroform solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, and then evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column with a volume ratio of 5:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 5:1), eluent containing the compound shown in the formula (I3 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 a), is obtained after drying, the yield is 62 percent, and the melting point is 221-223 ℃. 1 H NMR、 13 C NMR and HRMS were the same as in example 25.
Example 30: preparation of Compound (I3 b)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.573g (10.0 mmol) of compound (III 3 b) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column in a volume ratio of 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 b) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 b), is obtained after drying, the yield is 52 percent, and the melting point is 295-297 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.28(s,1H),8.83(s,1H),8.69(s,1H),8.60(s,1H),7.75(s,1H),7.21-7.12(m,5H),6.91(d,J＝7.9Hz,1H),6.55(dd,J＝8.2,2.2Hz,1H),3.73(s,3H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ160.1,155.1,152.7,149.5,141.7,141.2,140.2,139.2,129.9,129.2,113.9,113.3,110.8,110.0,107.6,104.3,55.3,18.0,15.9.HRMS(ESI)m/z[M+Na] + calcd for C 19 H 21 N 7 NaO 3 :418.1604,found:418.1600.
Example 31: preparation of Compound (I3 c)
2.968g (10.0 mmol) triphosgene is dissolved in 40mL of toluene, 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine is added dropwise under the condition of magnetic stirring at 0 ℃, the reaction solution is heated to room temperature after the dropwise addition, the reaction is carried out for 8 hours at the room temperature (TLC tracking detection is adopted in the reaction process, a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1 is used as a developing agent), nitrogen is introduced into the reaction system, and a tail gas absorption device is used for absorbing gas (the tail gas absorption liquid is 10 NaOH aqueous solution), until no white fog is generated in the tail gas absorption liquid, and the generated product is directly used for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 3 c) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column in a volume ratio of 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 c) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 c), is obtained after drying, the yield is 60 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.22(s,1H),8.78(s,1H),8.48(s,1H),8.41(s,1H),7.46(dd,J＝8.7,3.2Hz,2H),7.34(dd,J＝8.7,3.2Hz,2H),7.22(s,1H),7.15(d,J＝8.5Hz,1H),7.01(dd,J＝8.6,3.1Hz,1H),2.20(s,3H),2.16(s,3H),2.12(s,3H),1.93(s,3H).
Example 32: preparation of Compound (I3 d)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.873g (10.0 mmol) of compound (III 3 d) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL multiplied by 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after the solvent is evaporated, dissolving the residue to obtain a dissolved solution, then adding 4.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture is 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown as the formula (I3 d) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 d) is obtained after drying, the yield is 74 percent, and the melting point is 203-205 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.28(s,1H),8.82(s,1H),8.61(s,1H),8.43(s,1H),7.73(s,1H),7.20(s 1H),7.17-7.14(m,2H),6.88-6.81(m,2H),3.72(s,3H),3.71(s,3H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ155.0,152.8,149.4,149.1,144.3,141.6,140.3,139.1,133.6,129.0,113.6,113.1,112.8,110.4,109.8,104.2,56.1,55.6,17.9,15.7.HRMS(ESI)m/z[M+Na] + calcd for C 20 H 23 N 7 NaO 4 :448.1709,found:448.1706.
Example 33: preparation of Compound (I3 e)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.632g (10.0 mmol) of compound (III 3 e) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL × 3) water, separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after the solvent is evaporated, dissolving the residue to obtain a dissolved solution, adding 3.5 g of silica gel (300-400 mesh crude pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 e) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 e) is obtained after drying, the yield is 67 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.29(s,1H),9.07(s,1H),8.85(s,1H),8.45(s,1H),8.10(d,8.7Hz,1H),7.78(s,1H),7.36-7.28(m,1H),7.18-7.14(m,3H),7.05(t,J＝8.7Hz,1H),2.20(s,3H),1.95(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ154.6,152.3,149.0,141.2,140.0,138.7,137.2,136.2,129.5,129.3,128.7,119.4,115.6,113.2,112.6,109.3,17.6,15.4.
Example 34: preparation of Compound (I3 f)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.413g (10.0 mmol) of compound (III 3 f) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the mixture to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture to 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 f) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 f), is obtained after drying, the yield is 56 percent, and the melting point is 267-269 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.27(s,1H),8.80(s,1H),8.64(s,1H),8.46(s,1H),7.75(s,1H),7.36-7.29(m,2H),7.23-7.01(m,5H),2.24(s,3H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ163.4,155.0,149.4,141.4,140.1,139.4,137.0,135.3,132.4,129.4,129.3,123.2,122.6,121.4,119.4,118.0,20.6,17.9,15.7.
Example 35: preparation of Compound (I3 g)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.413g (10.0 mmol) of compound (III 3 g) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the mixture to obtain a dissolved solution, then adding 3.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture to 4:1 is used as eluent to elute, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 g) is collected according to TLC detection, the solvent of the collected eluent is evaporated, and the white solid product, namely the compound (I3 g) is obtained after drying, the yield is 60 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ9.27(s,1H),9.07(s,1H),8.81(s,1H),7.87(s,1H),7.86(s,1H),7.79(t,J＝7.1Hz,1H),7.27-7.22(m 1H),7.21-7.04(m,4H),6.93(td,J＝7.4,1.3Hz,1H),2.24(s,3H),2.19(s,3H),1.94(s,3H)； 13 C NMR(125MHz,DMSO-d 6 )δ155.0,152.6,149.3,141.6,140.2,139.7,139.1,137.9,129.0,123.6,116.1,113.5,113.0,109.6,21.3,17.9,15.7.
Example 36: preparation of Compound (I3 h)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.413g (10.0 mmol) of compound (III 3 h) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the mixture to obtain a dissolved solution, then adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the volume ratio of the mixture is 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 h) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 h) is obtained after drying, the yield is 73 percent, and the melting point is more than 300 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ8.43(s,1H),7.66(s,1H),7.27-7.23(m,4H),7.15(d,J＝7.6Hz,1H),7.05(d,J＝7.4Hz,1H),6.96(d,J＝7.4Hz,1H),6.86(s,1H),6.79(s,1H),6.68(s,1H),2.34(s,3H),2.33(s,3H),2.04(s,3H).
Example 37: preparation of Compound (I3 i)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 3 i) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 12 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL × 3) water, separating an organic phase, evaporating the solvent, carrying out column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after the solvent is evaporated, dissolving the residue to obtain a dissolved solution, adding 3.0 g of silica gel (300-400 mesh crude pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and the silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown as the formula (I3 i) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I3 i) is obtained after drying, the yield is 57 percent, and the melting point is 257-259 ℃. 1 H NMR(500MHz,DMSO-d 6 )δ8.41(s,1H),7.65(s,1H),7.23(s,1H),7.22(s,1H),7.01(d,J＝8.6Hz,2H),6.97(s,1H),6.89(s,1H),7.76(d,J＝8.6Hz,2H),6.59(s,2H),2.32(s,3H),2.29(s,6H),2.04(s,3H).
Example 38: preparation of Compound (I1 a)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 1.502g (10.0 mmol) of compound (III 1 a) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting at room temperature for 9 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), washing the reaction solution with (50 mL multiplied by 3), separating out an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation, dissolving the mixture to obtain a dissolved solution, then adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then mixing the mixture in a volume ratio of 1:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 1:1), eluent containing the compound shown as the formula (I1 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I1 a), is obtained after drying, the yield is 70 percent, and the melting point is 198-200 ℃. 1 H NMR as in example 2.
Example 39: preparation of Compound (I2 a)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.553g (10.0 mmol) of compound (III 2 a) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 10 hours (the reaction process is detected by TLC tracking, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 6:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 3.5 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column with a volume ratio of 6:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 6:1), eluent containing the compound shown in the formula (I2 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and the white solid product, namely the compound (I2 a), is obtained after drying, the yield is 57 percent, and the melting point is more than 300 ℃. 1 H NMR and 13 c NMR was the same as in example 13.
Example 40: preparation of Compound (I3 a)
Dissolving 2.968g (10.0 mmol) of triphosgene in 40mL of toluene, dropwise adding 20mL of toluene solution containing 1.121g (10.0 mmol) of 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine (II) and 1.012g (10.0 mmol) of triethylamine under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise adding, reacting for 8 hours at room temperature (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1), introducing nitrogen into the reaction system, absorbing gas by using a tail gas absorbing device (the tail gas absorbing solution is 10 NaOH aqueous solution), and generating no white fog in the tail gas absorbing solution, wherein the generated product is directly used for the next reaction.
Dissolving 2.273g (10.0 mmol) of compound (III 3 a) and 1.012g (10.0 mmol) of triethylamine in 40mL of toluene, dropwise adding the reaction solution obtained in the previous step under the condition of magnetic stirring at 0 ℃, heating the reaction solution to room temperature after dropwise addition, reacting at room temperature for 11 hours (the reaction process adopts TLC tracking detection, a developing agent is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 4:1), washing the reaction solution with (50 mL × 3), separating an organic phase, evaporating the solvent, performing column chromatography on the residue, namely adding 10mL of ethyl acetate solvent into the residue after solvent evaporation to dissolve the residue to obtain a dissolved solution, adding 4.0 g of silica gel (300-400 mesh coarse pore (zcx.II) type column chromatography silica gel into the dissolved solution, uniformly mixing, evaporating the solvent to obtain a mixture of dried residue and silica gel, loading the mixture into a column, and then loading the mixture into the column at a volume ratio of 4:1 is used as eluent, elution is carried out, TLC tracking detection is carried out (the developing solvent is mixed solution of petroleum ether and ethyl acetate with the volume ratio of 4:1), eluent containing the compound shown in the formula (I3 a) is collected according to TLC detection, the solvent is evaporated from the collected eluent, and a white solid product, namely the compound (I3 a), is obtained after drying, the yield is 75 percent, and the melting point is 221-223 ℃. 1 H NMR、 13 C NMR and HRMS were the same as in example 25.
Example 41: in vitro kinase Activity assay
Kinase-Lumi TM A chemical luminescence method kinase activity detection reagent kit is a reagent kit for quantitatively detecting the kinase activity by measuring the residual amount of ATP in a solution after kinase reaction by a chemical luminescence method. Kinase-Lumi is adopted in the experiment TM Chemiluminescence method kinase activity assay kit (Beyotime) was used to test the inhibitory activity of the compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d), (I3 e) prepared in the above examples on EGFR, PEGFR β and VEGFR-2 kinase, respectively, at room temperature, wherein the compound (I2 a) was prepared by the method of example 39.
The following is a 96-well plate recommended detection system.
(1) Preparation of ATP Standard Curve
The reaction buffer was prepared with 1mM manganese dichloride, 5mM magnesium dichloride, 1mM Dithiothreitol (DTT).
Set 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μ MATP standard wells (all ATP concentrations above are final concentrations of the substance when the total volume in the standard wells reaches 100 μ L). For preparation, 50. Mu.L of ATP was first diluted with reaction buffer. Then 50 mu Lkinase-Lumi is added TM And (3) mixing the chemiluminescence kinase detection reagent and the mixture. After reaction at room temperature (about 25 ℃) for 10 minutes, chemiluminescence detection was carried out using a multifunctional microplate reader, and an ATP standard curve was prepared.
(2) Sample detection
The sample wells were configured to contain 0.1. Mu.g/mL polyglutamic acid and tyrosine (4:1) kinase substrates, 5. Mu. MATP and 10. Mu.g/L kinase (EGFR, PEGFR. Beta. Or VEGFR-2), and different concentrations (50, 100, 200, 400, 800, 1000 nM) of compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d), (I3 e) when the total volume per well reached 100. Mu.L. When the reagent is prepared, polyglutamic acid and tyrosine (4:1) kinase substrate, ATP, kinase (EGFR, PEGFR beta or VEGFR-2) and compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d) and (I3 e) are added into each hole, and reaction buffer solution is added to dilute the mixture until the total volume is 50 mu L; then 50 mu Lkinase-Lumi is added TM And (3) mixing the chemiluminescence kinase detection reagent and the mixture. The reaction was carried out at room temperature (about 25 ℃ C.) for 10 minutes. Then using a multifunctional microplate reader to perform chemiluminescence detection. The amount of ATP remaining in the sample wells was calculated from the standard curve, and then the enzyme activity was calculated according to the definition of enzyme activity. Finally calculate the IC 50 The value is obtained.
In vitro kinase Activity assay results are shown in Table 1, which shows the activity data of compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d), (I3 e) on EGFR, PEGFR β and VEGFR-2 kinase. It can be seen from the data in the table that the compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d), (I3 e) have different degrees of inhibitory effect on the EGFR, PEGFR beta and VEGFR-2 activities, and that the inhibitory activity of the compounds (I1 e), (I1 f), (I2 a), (I2 b), (I2 c), (I2 d), (I2 e), (I3 d) and (I3 e) on VEGFR-2 kinase is superior to that of EGFR and PDGFR beta as a whole.
TABLE 1 tyrosine kinase inhibitory Activity of Compound (I)
Example 42: in vitro test for anti-cancer Activity
The compounds (i) prepared in the above examples were tested for biological activities of human lung cancer cell a549, human hepatoma cell Huh7, human hepatoma cell HepG2 and human breast cancer cell MDA-MB-231, respectively, wherein the compounds (i 1 a), (i 2 a) and (i 3 a) were prepared by the methods of example 38, example 39 and example 40, respectively.
The test method comprises the following steps: tetrazolium salt reduction (MTT process).
Cell lines: human lung cancer cell A549, human liver cancer cell Huh7, human liver cancer cell HepG2 and human breast cancer cell MDA-MB-231. The tumor cell strain is purchased from cell banks of Shanghai Life sciences of Chinese academy of sciences.
The experimental procedure was as follows:
(1) Culture of tumor cells
The cell generation number used in the experiment is within 5 generations. The consumables and reagents used for cell culture are subjected to rigorous sterilization procedures, and all experimental procedures are performed in a sterile operating station.
(a) Resuscitation of tumor cells
Before the experiment is started, articles to be used, such as a culture bottle, a centrifuge tube, a pipetting gun, a gun head, a waste liquid barrel and the like, are placed in a super clean bench, an ultraviolet lamp is turned on for sterilization, and reagents used for cell culture are placed in a constant-temperature water bath kettle at 37 ℃ for preheating. The ultraviolet lamp is turned off after all the work is ready,and (5) starting the super-clean bench fluorescent lamp and the ventilating fan. Taking out the frozen tumor cells from a refrigerator at the temperature of minus 80 ℃, shaking the cells in a water bath at the temperature of 37 ℃ for quick thawing, spraying alcohol on the cells to place the cells in a super clean bench under the condition that a small part of frozen liquid in the frozen tube is still unfrozen, immediately transferring all the cells in the frozen tube to a 15mL centrifuge tube added with 1640 culture solution, and lightly blowing and uniformly mixing the cells by using a pipette gun. Placing the centrifugal tube in a centrifuge, centrifuging at 1000rpm for 5min, and sucking off the supernatant in a super clean bench. Sucking 2mL1640 culture solution into a centrifuge tube containing cell sediment by using a pipette to prepare cell suspension, blowing, beating, uniformly mixing and transferring the cell suspension to a bottle with the bottom area of 25cm 2 The air-permeable culture flask of (3) was supplemented with 1640 medium (4 mL), the cells were mixed by gently shaking the flask, and the flask was charged to 5% CO 2 And culturing in a constant temperature incubator at 37 ℃.
(b) Passage of tumor cells
When the cell growth state is good and the bottom of the culture bottle is 70% -80%, the cell can be subcultured. The passage operation steps are all completed in a super clean bench, and the passage can be started after the preparation work is done. Firstly, sucking the original culture solution in a culture bottle off by a pipette gun and beating the culture solution into a waste solution barrel, adding 2mL of PBS for repeated washing for several times, then adding about 600-700 mu L of trypsin (containing 0.02% of EDTA, phenol red and 0.25% of pancreatin) to ensure that the bottom of the bottle is completely covered by the trypsin, slightly shaking the culture bottle, putting the culture bottle into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, and slightly beating the bottle wall by using a finger belly if a small amount of cells are still attached to the wall until most of the cells can shed from the bottom of the bottle. After the digestion was terminated by adding 2mL of 1640 medium, the cells were gently pipetted using a pipette gun, the cells were completely blown off the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. Aspirating the supernatant, diluting with 1640 medium, beating homogeneously, distributing into 2-3 flasks in average, and further processing with 5% CO 2 And culturing in a constant-temperature incubator at 37 ℃.
(c) Cryopreservation of tumor cells
A cell freezing solution was prepared in advance in a volume ratio of FBS (fetal bovine serum) to DMSO =9:1, and placed in a refrigerator at 4 ℃ for use. And (4) freezing and storing operation steps are finished in the super clean bench, and freezing and storing can be started after preparation work is done. Firstly, sucking the original culture solution in a culture bottle, adding 2mL of PBS buffer solution for repeated washing for several times, pouring out, then adding about 600-700 mu L of trypsin (containing 0.02 percent of EDTA, phenol red and 0.25 percent of pancreatin), slightly shaking the culture bottle to ensure that the pancreatin can cover the bottom of the culture bottle, putting the culture bottle into a constant-temperature incubator at 37 ℃ for incubation for 1-2 min, observing the cell shedding condition under a microscope, and slightly knocking the bottle wall by using a finger belly if a small amount of cells are still attached to the wall until most of the cells can shed from the bottle bottom. After the digestion was terminated by adding 2mL of 1640 medium, the cells were gently pipetted using a pipette gun, the cells were completely blown off the bottom of the flask, and the cell suspension was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. The supernatant was decanted off. 1mL of the cryopreservation solution just taken out of the refrigerator at 4 ℃ was added, and the mixture was blown up uniformly to form a cell suspension, which was transferred into a cryopreservation tube. Standing at 4 deg.C for 30min after labeling cell type, cell generation number and freezing storage date, standing at-20 deg.C for 1 hr, and storing in-80 deg.C ultra-low temperature refrigerator.
(2) MTT experimental method
(a) Cell counting: digesting and centrifuging tumor cells with good growth state in a culture flask, then re-suspending the tumor cells by using 4mL1640 culture solution, taking 10 mu L of cell suspension to a cell counting plate, counting, diluting B16F10 cells to 5 multiplied by 10 4 one/mL.
(b) Plate paving: taking a 96-well plate, adding 100 mu L of diluted cell suspension into an experimental well, adding 100 mu L of 1640 culture solution into a blank well, supplementing 100 mu L of LPBS buffer solution around the blank well, and adding CO 2 Culturing in a constant temperature incubator.
(c) Preparing a compound and adding medicine: the compound prepared by DMSO in 10. Mu. Mol/mL and the positive control drug Sorafenib were diluted to the specified concentrations in 1640 medium at 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, and 1.25. Mu.M, respectively, and the drug delivery was carried out by the solution change method. Discarding the original culture solution, adding 100 μ L1640 culture solution containing compound or positive control drug into the experimental well, adding 100 μ L1640 culture solution into the control group and blank well, adding the drug, and adding the 96-well plate into 5% CO 2 And continuously culturing in a constant-temperature incubator at 37 ℃.
(d) Adding MTT: after 48 hours, the 96-well plate was taken out and placed in a clean bench, and 10. Mu.L of 5mg/mL MTT solution was added to each well in the dark, followed by 5% CO 2 And continuously culturing in a constant-temperature incubator at 37 ℃.
(e) And (3) detection: the 96-well plate added with MTT is incubated in the incubator for 3.5-4 h and taken out, the solution in each well is carefully sucked out, 150 mu L of DMSO is added into each well to dissolve generated formazan, then the formazan is placed on a flat plate oscillator to vibrate for 20min, and an enzyme-labeling instrument is used for detecting the absorbance value under 490 nm.
(f) And (3) processing experimental data: the cell viability was calculated according to the following formula, and the value of 50% of the cell viability was IC 50 。
Cell survival (%) = [ (As-Ab)/(Ac-Ab) ]. Times.100%
As assay well (containing cell culture solution, MTT, toxic substance)
Ac control well (cell culture, MTT, no toxic substance)
Ab blank well (MTT-containing, cell and toxic free medium).
The results of the tests are shown in table 2:
TABLE 2 inhibitory Effect of Compound (I) on cancer cell growth
Claims (10)
1. A diamethymetrizine compound is characterized in that: the diformylamido-symtetrazine compound has the following structural formula (I):
in the formula (I), R is methyl, ethyl, propyl, isobutyl, benzyl, 3-chlorobenzyl, 3,4-dimethoxybenzyl, 3-methylphenylaminomethyl, 3,4-dimethylphenylaminomethyl, 3-trifluoromethyl-4-chlorophenylaminomethyl, 3,4-dichlorophenylaminomethyl, 3-fluoro-4-chlorophenylaminomethyl, phenylaminomethyl, 2-methylphenylaminomethyl, 3,5-dimethylphenylaminomethyl, phenylamino, 3-methoxyphenylamino, 3,4-dimethoxyphenylamino, 2,5-difluorophenylamino, 2-methylphenylamino, 3-methylphenylamino, 4-methylphenylamino or 3,5-dimethylphenylamino.
2. A process for the preparation of a bis-methylamido-tetrazine compound as claimed in claim 1, wherein: the preparation method comprises the following steps:
(1) Adding triphosgene into an organic solvent A, dropwise adding an organic solvent A solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in a formula (II) and a basic catalyst a under the stirring condition of-10-12 ℃, heating reaction liquid to room temperature after the dropwise adding is finished, stirring and reacting for 0.5-50 hours at room temperature, introducing nitrogen into a reaction system until tail gas absorption liquid does not generate white mist, and directly using a generated product for next reaction;
(2) Adding a compound shown in a formula (III) and a basic catalyst B into an organic solvent B, stirring for dissolving, dropwise adding the reaction liquid obtained in the step (1) at the temperature of-10-12 ℃ under the stirring condition, heating the reaction liquid to room temperature after the dropwise adding is finished, stirring for reacting for 0.5-50 hours at room temperature, and then separating and purifying the reaction liquid to obtain the dimethylamido-sym-tetrazine compound shown in the formula (I);
the basic catalyst a and the basic catalyst b are one of the following: triethylamine, 4-dimethylaminopyridine, pyridine or sodium hydroxide;
in the formula (III), R is as defined in the formula (I).
3. The method of claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the alkaline catalyst a, the amount of the alkaline catalyst b, the amount of the triphosgene and the amount of the compound (III) to be fed is 1: 0.1 to 3.
4. The method of claim 2, wherein: the ratio of the amount of the compound (II) to the amount of the alkaline catalyst a, the amount of the alkaline catalyst b, the amount of the triphosgene and the amount of the compound (III) to be fed is 1: 0.1 to 2.
5. The method of claim 2, wherein: the organic solvent A and the organic solvent B are respectively and independently selected from one of the following: dichloromethane, chloroform or toluene.
6. The method of claim 2, wherein: in the step (1), under the condition of stirring at the temperature of between 10 ℃ below zero and 5 ℃, dropwise adding an organic solvent A solution containing 3,6-dimethyl-1,6-dihydro-1,2,4,5-tetrazine shown in a formula (II) and a basic catalyst a;
in the step (2), the reaction solution obtained in the step (1) is dropwise added under the condition of stirring at a temperature of between 10 ℃ below zero and 5 ℃.
7. The use of a bis-methylamido-tetrazine compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, in the manufacture of a medicament for treating or preventing VEGFR-2 mediated diseases or inhibiting VEGFR-2, wherein the VEGFR-2 mediated disease is cancer and the cancer cells of the cancer are a549, huh7, MDA-MB-231 or HepG2 cells.
8. The use of claim 7, wherein: the cancer cells are A549 cells, and the diformylamido-homotetrazine compound is a compound (I2 a), (I2 b), (I2 c), (I2 d), (I2 e) or (I2 g);
9. the use of claim 7, wherein: the cancer cells are Huh7 cells, and the diformylamido-homotetrazine compound is a compound (I2 b), (I2 c), (I2 e), (I3 d) or (I3 i);
10. the use of claim 7, wherein: the cancer cell is HepG2 cell, the diamido-tetrazine compound is a compound (I2 b), (I2 c) or (I3 d);
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102267983A (en) * | 2011-06-16 | 2011-12-07 | 台州职业技术学院 | Sym-triazine derivative compounds containing sym-tetrazine rings and preparation method thereof |
| CN104098524A (en) * | 2014-05-12 | 2014-10-15 | 浙江工业大学 | 1-m-methoxy benzoyl-3-phenyl-1, 4-dihydro-1,2,4,5-tetrazine and preparation and application thereof |
| CN105968064A (en) * | 2016-05-06 | 2016-09-28 | 浙江工业大学 | Bis(m-methylphenyl) tetrazine dicarboxamide compound as well as preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102267983A (en) * | 2011-06-16 | 2011-12-07 | 台州职业技术学院 | Sym-triazine derivative compounds containing sym-tetrazine rings and preparation method thereof |
| CN104098524A (en) * | 2014-05-12 | 2014-10-15 | 浙江工业大学 | 1-m-methoxy benzoyl-3-phenyl-1, 4-dihydro-1,2,4,5-tetrazine and preparation and application thereof |
| CN105968064A (en) * | 2016-05-06 | 2016-09-28 | 浙江工业大学 | Bis(m-methylphenyl) tetrazine dicarboxamide compound as well as preparation and application thereof |
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